This study is intended to elicit and utilize basic biochemical information concerning the enzymes metabolizing pyruvate for developing methods of detection of genetic diseases affecting these enzymes and for studying the physiological, biochemical and clinical effects of such inborn errors. The studies will continue to concentrate on pyruvate dehydrogenase complex and pyruvate carboxylase, the two major enzymes of pyruvate utilization. Defects in pyruvate dehydrogenase complex appear to be responsible for a significant number of diseases involving lactic acidosis, neurological problems and mental and developmental retardation. Assays are now available for detecting some but not all of the different types of defects of this complex. We plan to develop general screening procedures to detect this set of disorders. In addition, we hope to be able to classify the errors into specific biochemical lesions. The regulation of pyruvate dehydrogenase in normal tissues is still not completely understood, particularly the role played by multi-site phosphorylation. The latter process will be studied in purified enzymes, mitochondria and whole organs and the knowledge used in the development of the assays mentioned above. The metabolism of alpha-ketobutyrate, a naturally occurring analog of pyruvate, will be examined as a potential means of studying pyruvate metabolism in vivo. Several cases of pyruvate carboxylase deficiency have already been detected. Present plans are to investigate the nature of the biochemical lesion in these cases using fibroblasts which are deficient in the enzyme. Other studies will involve NADPH dehydrogenase deficiency, a defect also leading to lactic acidosis. Finally, studies with stable isotopes will attempt to elucidate the metabolic effects of diseases involving pyruvate, oxidative and carbohydrate metabolism in children.